We seek to develop and commercialize a powerful technology platform to increase dramatically the effectiveness of drug-discovery using high-throughput screening (HTS) based on fluorescence lifetime (FLT) readouts of FRET in live cells. This breakthrough is enabled by a combination of two complementary and synergistic technologies: fluorescent biosensor engineering (University of Minnesota, UMN) and fluorescence instrumentation engineering (Fluorescence Innovations, FI). In Phase I we achieved our aims, producing the first truly high-throughput and high-precision applications of FLT in living cells, applied to a specific biosensor (SERCA). We also surpassed our aims by developing new instrumentation for high-throughput spectral recording, carrying out a SERCA screen on a 50,000-compund library, and developing FRET biosensors based on several new targets. In addition, several pharmaceutical companies expressed interest in this technology platform, so UMN and FI started a new drug-discovery company, Photonic Pharma, for the ultimate commercialization of these combined technologies. In Phase II, we will further develop this technology platform and apply it to a diverse array of targets, thus clearly demonstrating the high potential for commercialization. In In Aim 1 we will seek improvements in hardware and software that will enhance future commercialization potential, including a new instrument that combines FLT and spectral recording, increased digitizer speed, higher density plates, connectivity with robotics, and software improvements. In Aim 2, in order to demonstrate that our technology platform is widely applicable, we will expand our list of biosensors, focusing on targets of high commercial potential, for which world-leading experts are in close proximity at UMN. These disease targets include heart failure, drug abuse and addiction, inflammation and cancer, and muscle dystonia. Biosensors will be engineered and expressed in live cells, and screened against standard test and validation libraries to optimize the screening assay. We will then carry out several large-scale (50,000-compound) screens, using optimized HTS assays. Hit compounds will be retested as a function of compound concentration (dose-response), and then subjected to functional assays performed by the above world-class experts at UMN. Promising hits may become leads for future medicinal chemistry development, but the primary goal is to validate the discovery platform. We are confident that the research conducted under these aims will set us up nicely for the commercialization phase by validating our business plan to combine UMN expertise in biosensor engineering and cell culture with FI expertise in instrumentation, led by Photonic Pharma, an emerging early-phase drug discovery start-up company. Thus our business plan is not to sell instruments, but to leverage our unique combination of biosensor and instrumentation expertise to develop and apply a technology platform that accelerates the early phase of drug discovery.